CN104152491A - Fermented Radix Astragali preparation method, and method for extracting total saponins of fermented Radix Astragali - Google Patents
Fermented Radix Astragali preparation method, and method for extracting total saponins of fermented Radix Astragali Download PDFInfo
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Abstract
The invention provides a fermented Radix Astragali preparation method. Fermented Radix Astragali is obtained through fermenting Radix Astragali by Streptococcus alactolyticus strains LZMYFGM9 with the preservation number of CGMCC No.4227. The invention also provides a method for extracting total saponins of fermented Radix Astragali. The extraction method comprises the following steps: 1, carrying out warm immersion extraction on the fermented Radix Astragali by 80-90% ethanol, preferably 85% ethanol in order to obtain a fermented Radix Astragali alcohol extract fluid extract; 2, extracting the fermented Radix Astragali alcohol extract fluid extract obtained in step 1 by water saturated n-butanol in order to obtain an extract liquid; and 3, adding the extract liquid into a macro-porous adsorption resin column for absorption, euting, collecting the obtained eluate, and post-processing the eluate to obtain purified total saponins of the fermented Radix Astragali. The purity of saponins is further improved through the extraction method, and reaches 86-92%.
Description
Technical field
The invention belongs to biotechnology and medical technical field, the preparation method of the Radix Astragali and the extracting method of total saponins thereof are specifically related to ferment.
Background technology
The Radix Astragali (Radix Astragalus) is the dry root of leguminous plants Radix Astagali or Radix Astragali.Taste is sweet, and slightly warm in nature has effect of beneficial gas qi-restoratives.Radix Astragali total saponins comprises tens kinds of saponins materials, has the effects such as antibacterial, anti-inflammatory, antitumor, hypotensive, anti-myocardial anoxia, central analgesia, calmness, is the main effective constituent of the Radix Astragali, and its content in crude drug is 0.095 %～0.223 %.Because content is lower, therefore, researchist continually develops the high efficiency separation extractive technique of Radix Astragali total saponins on the one hand, is devoted on the other hand to be synthesized and had bioactive Radix Astragali saponin by the method for bio-transformation.
Microbial transformation refers to by microorganism whole cell or enzyme system carries out structural modification by complicated substrate, and this catalyzed reaction has mild condition, reacts single-minded, pollutes the features such as little.Set up after rational Chinese medicine microbial transformation technique, can be in obtaining the target product that yield is higher, other effective constituent in comprehensive utilization medicine, conservation greatly, reduces production costs, and reaches the object of comprehensive utilization active ingredients from traditional Chinese medicinal.The method of conventional microbial transformation Effective Component of Chinese Medicine has solution fermentation and solid fermentation method.
Non-solution lactose suis LZMYFGM9(deposit number: CGMCC No.4227) secretion a lot of enzymes can participate in biotransformation, but, apply this bacterium fermentation Radix Astragali, in the Radix Astragali, the variation of total saponins it be not immediately clear, for the separating and extracting method of the saponin(e of this bacterium of application fermentation Radix Astragali, there is not yet and studies have reported that.
preservation information:
The Latin Classification And Nomenclature of non-solution lactose suis LZMYFGM9 is
streptococcus alactolyticus;
Preservation date: on October 19th, 2010;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Depositary institution is called for short: CGMCC;
Depositary institution address: No. 3, No. 1, BeiChen West Road, Chaoyang District, BeiJing City institute;
Deposit number: CGMCC No.4227.
Summary of the invention
Technical problem to be solved by this invention is to propose a kind of economy, the ferment preparation method of the Radix Astragali and the extracting method of total saponins thereof efficiently.
For achieving the above object, the technical solution used in the present invention is as follows:
The preparation method who the invention provides a kind of Radix Astragali that ferments, step is as follows: after clean Radix Astragali crude drug medicine materical crude slice is pulverized, the fermentation Radix Astragali, the bacterial concentration of fermentation strain CGMCC No.4227 is 2 × 10
6～2 × 10
8individual/mL, connecing bacterium amount is that 4%～5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 38 DEG C～39 DEG C of temperature, rotating speed 200r/min, pH value 5.5～6.5, dissolved oxygen amount 5%～10%, neutralizing agent is the NaOH of 6mol/L, fermentation time 44h～52h, after fermentation ends, the freeze-drying fermentation astragalus liquid Radix Astragali product that must ferment; Preferably, the bacterial concentration of fermentation strain CGMCC No.4227 is 2 × 10
8individual/mL, connecing bacterium amount is that 5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 39 DEG C of temperature, rotating speed 200r/min, pH value 6.4, dissolved oxygen amount 10%, the NaOH that neutralizing agent is 6mol/L, fermentation time 48h.
Above-mentioned fermentation condition is the optimum value obtaining after orthogonal test.
Preparation method of the present invention has the following advantages:
Radix Astragali zymotechnique in the present invention uses 10L tri-fermentor tanks, production operation is easy, easy control of reaction conditions (can realize in fermenting process and automatically regulate in real time pH value, oxygen dissolving value, the parameter such as temperature, rotating speed also can reach accurate control), thereby leavened prod quality is more stable, differences between batches are little.Meanwhile, present method industrialization degree is high, and production cost is low, has improved the utilization ratio of raw material, has strengthened the competitiveness of product in market.
Preferably, the liquid nutrient medium using in described fermentation Radix Astragali process has been prepared by the composition of following parts by weight: whey powder: 24～27 parts; Peptone: 2.2～2.4 parts; Glucose: 0.5～0.7 part; Yeast powder: 1.4～1.7 parts; Magnesium sulfate: 0.03～0.05 part; Triammonium citrate: 0.30～0.50g; Hydrogen sulfate dipotassium: 0.30～0.50g; Sodium acetate: 0.90～1.10g; Astragalus membranaceus powder: 80～100 parts; Water: 900～1100 parts.
Experimental studies have found that, the normal growth of fermentation strain CGMCC No.4227 needs the energy, carbon source, nitrogenous source, mineral element, water and somatomedin etc., the substratum that laboratory adopts is in the past the substratum described in " a kind of fermention medium that extracts astragalus polysaccharides " (201210141832.6), wherein, inorganic salt are potassium primary phosphate and dipotassium hydrogen phosphate, lack the nitrogen sources such as main mineral elements and inorganics ammonium salt such as Na, Mg.The present invention adds after above-mentioned nutritive ingredient, simultaneously, nutrient media components is optimized in conjunction with experiment of single factor and response surface analysis, draws substratum best composition and each component ratio, use the growth conditions of this substratum fermentation strain CGMCC No.4227 obviously to improve.
More preferably, the liquid nutrient medium using in described fermentation Radix Astragali process has been prepared by the composition of following parts by weight: whey powder: 25.79 parts; Peptone: 2.28 parts; Glucose: 0.60 part; Yeast powder: 1.56 parts; Magnesium sulfate: 0.04 part; Triammonium citrate: 0.40 part; Hydrogen sulfate dipotassium: 0.40 part; Sodium acetate: 1.00 parts; Astragalus membranaceus powder: 80 parts; Water: 1000 parts.
The present invention also provides the application of the above-mentioned fermentation Radix Astragali as fodder additives.
The 3rd object of the present invention is to provide a kind of fodder additives, and the effective constituent of described fodder additives is the fermentation Radix Astragali claimed in claim 1.
The 4th object of the present invention is to provide a kind of extracting method of the Radix Astragali total saponins that ferments, and step is as follows:
1) the above-mentioned fermentation Radix Astragali is got with 80%～90% ethanol temperature lixiviate, preferably got with 85% ethanol temperature lixiviate, obtain alcohol extracting fermentation Radix Astragali fluid extract; Preferably, in described fluid extract, 1g fluid extract contains 0.9～1.1g crude drug Radix Astragali.
2) alcohol extracting fermentation Radix Astragali fluid extract water saturation n-butanol extraction step (1) being obtained, obtains extracting solution;
3) extracting solution is splined on to macroporous adsorptive resins absorption, wash-out, collects elutriant, and elutriant is carried out to aftertreatment, obtains the fermentation Radix Astragali total saponins after purifying.
Preferably, it is that 83 DEG C～86 DEG C temperature are soaked 1h～2h by airtight immersion 6h～10h under 80%～90% ethanol room temperature for the fermentation Radix Astragali that described temperature lixiviate is got, and filters, and filter residue repeats to extract 1 time again; Preferably, it is that 85 DEG C of temperature are soaked 1h by airtight immersion 8h under 85% ethanol room temperature for the fermentation Radix Astragali that described temperature lixiviate is got, and filters, and filter residue repeats to extract 1 time again.
When temperature lixiviate is got middle selection above-mentioned parameter and extracted, extraction yield is the highest.
Preferably, when described temperature lixiviate is got, every 1g fermentation Radix Astragali 20ml 85% ethanol temperature lixiviate is got.
This ratio is that experiment of single factor optimizes, the Radix Astragali fluid extract yield of fermenting under these conditions and wherein total saponin content is the highest.
Preferably, while using water saturation n-butanol extraction in step (2), at the uniform velocity slight jolting in the same direction, after slightly static, slighter jolting in the other direction.
Preferably, described in step (3), macroporous adsorbent resin is AB-8 or D101; Be preferably AB-8.
Preferably, described in step (3), wash-out is first to use 30% ethanol elution, abandons elutriant; This polarity section material to macroporous resin adsorption power a little less than, be not total saponins class, therefore discard elutriant; Use again 70%～90%% ethanol elution, collect elutriant.This polarity section material is stronger to macroporous resin adsorption power, for fermentation Radix Astragali total saponins class, therefore collect elutriant.Preferably, described wash-out is first to use 30% ethanol elution, abandons elutriant; Use again 85% ethanol elution, collect elutriant.
Further, after extracting, elutriant is after concentrate drying, and fermentation Radix Astragali total saponins content reaches 78.52%～88.39%.
The present invention utilizes a strain to separate the non-solution lactose suis LZMYFGM9 from chicken enteron aisle, and deposit number is: CGMCC No.4227 and Chinese medicine astragalus ferment altogether, develops fermentation Radix Astragali product.This product can strengthen broiler growth performance as fodder additives, improves immunizing power.Saponin(e is one of main pharmacodynamics material of fermentation Radix Astragali product, research report, and in digestive tube, bacteriogenic enzyme selectivity ground decomposes saponin component, causes the transformation of structure, makes it be decomposed into aglycon and glycosyl.(as the people such as Kim utilize Vitamin C2-R2A to filter out 70 strains to show the bacterial strain of beta-glycosidase activity, wherein to transform Rb1 be Rd in 12 strains, wherein three strain bacterium
burkholeria pyrrociniagP16,
bacillus megateriumgP27 and
sphingomonas echinoidesgP50 can transform completely in 48 hours.Dong etc. have screened 49 kinds of microorganisms to investigate the conversion to ginsenoside Rg1, and experiment finds that it is Rh1 that aspergillus niger AS3.1858 and the mould AS3.3538 of blue colter can transform Rg1, and transformation efficiency is 80.9%.) because the polarity of aglycon is significantly less than the saponin(e before decomposition, so more easily by intestinal absorption and then enter blood, performance drug action.By microbial transformation, be expected to improve the content of the saponin(e that has drug action, produce new saponin(e kind, find new pharmacologically active.In addition, bio-transformation hydrolysis glycoside also has protection of the environment, and the advantage such as reduce production costs, is applied to suitability for industrialized production, and the development to medicinal industry is contributed.Therefore, fermentation Radix Astragali total saponins demonstrates good development prospect.
The present invention also extracts to fermentation Radix Astragali total saponins the technique separating and investigates, and adopts vanillin-sulfuric acid method, taking Cyclosiversioside F as standard substance, detects saponin content, has obtained the optimum extracting method of fermentation Radix Astragali total saponins.Meanwhile, also for the extraction of other liquid fermenting Chinese medicine saponin(e provides reference method.
The effect of extracting method of the present invention and benefit:
1, first soak the fermentation Radix Astragali by 85% ethanol temperature and obtain the molten extracting section total saponins of alcohol composition, the dregs of a decoction are used for extracting other effective constituent, are beneficial to abundant exploitation and efficient utilization of leavened prod effective constituent.
2, propyl carbinol method and Flavonoids by Macroporous Adsorption Resin are combined, further improved the purity of total saponins, reach 86 %～92 %.
3, through the purifying of propyl carbinol, the color of total saponins is very shallow, and recyclable propyl carbinol reuses, and has saved in macroporous resin column by the step of ether decolouring, has reached the effect economizing on resources.
4, the present invention compares by the concentration effect of the total saponins to macroporous resin AB-8 and D101, find that AB-8 is better than D101, have that total saponins extraction efficiency is high, purity high, and filler can be recycled, the wasting of resources is few, easy and simple to handle, be easy to promote and expanding production.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The mensuration of Radix Astragali total saponins content of fermenting in the present invention adopts vanillin-sulfuric acid method, and step is as follows:
The making of a, typical curve: with anhydrous alcohol solution Cyclosiversioside F standard substance, be mixed with the standard solution that concentration is 0.5 mg/mL.Draw respectively 0.15 mL, 0.3 mL, 0.45 mL, 0.6 mL, 0.75 mL to test tube, add dehydrated alcohol to 0.75 mL.Retinue is done blank with 0.75 mL dehydrated alcohol.Precision adds 8 % Vanillin-ethanol solution 0.75 mL, adds 72 % H in ice bath
2sO
47.5 mL, shake up, and are placed in after 62 DEG C of water-bath constant temperature leave standstill 20 min and take out.Ice bath is cooled to room temperature, shakes up.In 30 min in 544 nm places mensuration absorbancys, taking Cyclosiversioside F concentration as X-coordinate (x), absorbance (A) is ordinate zou (y), carries out linear regression, drawing standard curve.Typical curve equation is: y=1.3326x+0.0117 (R
2=0.9997), wherein: y is absorbance, x is Cyclosiversioside F concentration (mg/mL), R
2for relation conefficient;
B, total saponin content measure: the fermentation Radix Astragali total saponins making is made into the solution that concentration is 0.5 mg/mL with dehydrated alcohol, get 0.75 mL measures 544 nm absorbance by the method for step a, calculate the concentration of fermentation Radix Astragali total saponins according to typical curve;
C, fermentation Radix Astragali total saponins content (%)=(typical curve calculate saponin(e quality/purifying obtain fermenting Radix Astragali saponin quality) × 100%
Total saponin (%)=(purifying obtains the quality of quality/fermentation Radix Astragali product of total saponins) × 100% after d, fermentation Radix Astragali purifying.
The deposit number of the present invention non-solution lactose suis (Streptococcus alactolyticus) LZMYFGM9 used is CGMCC No.4227.The cultural characters of this bacterial classification is shown in Chinese patent 201210141827.5(denomination of invention: a kind of non-solution lactose suis and application thereof).
embodiment 1
The preparation method of the fermentation Radix Astragali of the present invention is:
After clean Radix Astragali crude drug medicine materical crude slice is pulverized, cross 20 mesh sieves, with the 10L ferment tank Radix Astragali, the bacterial concentration of fermentation strain LZMYFGM9 is 2 × 10
8individual/mL, connecing bacterium amount is that 5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 39 DEG C of temperature, rotating speed 200r/min, pH value 6.4, dissolved oxygen amount 10%, the NaOH that neutralizing agent is 6mol/L, fermentation time 48h.After fermentation ends, the freeze-drying fermentation astragalus liquid Radix Astragali lyophilized powder that must ferment.Recording in the fermentation Radix Astragali massfraction of the Radix Astragali and be in 78%, 400g fermentation Radix Astragali lyophilized powder is 312g containing the quality of the Radix Astragali.
The liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 25.79 parts; Peptone: 2.28 parts; Glucose: 0.60 part; Yeast powder: 1.56 parts; Magnesium sulfate: 0.04 part; Triammonium citrate: 0.40 part; Hydrogen sulfate dipotassium: 0.40 part; Sodium acetate: 1.00 parts; Astragalus membranaceus powder: 80 parts; Water: 1000 parts.
The Radix Astragali lyophilized powder that ferments in embodiment 1 is made an addition to according to 1% dosage in the broiler fodder of 21 ages in days and feed 28 days, after off-test, find that the body weight of fermentation Radix Astragali group broiler chicken is apparently higher than blank group and crude drug Radix Astragali group (P>0.05), the Radix Astragali group of fermenting as calculated average daily gain only reaches 34.25g/d, the average daily gain of blank group only reaches 30.15 30.49g/d, and crude drug Radix Astragali group only has 26.13g/d only; And the product A as a control group that the fermention medium in application patent 201210141832.6 is fermented according to fermentation process of the present invention, after feeding dorking, average daily gain is 31.69 g/d.Fermentation Radix Astragali group experimental chicken internal organs are cutd open to inspection and have no any pathological change, the heart of the Radix Astragali group of simultaneously fermenting, liver, renal index not remarkable (P>0.05) of difference compared with Normal group.Detect fermentation Radix Astragali group broiler chicken haematogenic immunity globulin levels, result shows, all increases to some extent of fermentation Radix Astragali group broiler chicken haematogenic immunity sphaeroprotein compared with blank group, and IgA content has improved 1.5%, IgM content and has improved 1.93 %, and IgG has improved 1.88%.And control group A is compared with blank group, IgA content has improved 1.3%, and IgM content has improved 1.85%, and IgG has improved 1.73%.This leavened prod of results suggest can strengthen broiler growth performance as fodder additives, and can improve immunity of organisms.
embodiment 2
The preparation method of the fermentation Radix Astragali of the present invention is:
After clean Radix Astragali crude drug medicine materical crude slice is pulverized, cross 20 mesh sieves, with the 10L ferment tank Radix Astragali, the bacterial concentration of fermentation strain LZMYFGM9 is 2 × 10
6individual/mL, connecing bacterium amount is that 4%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 38 DEG C of temperature, rotating speed 200r/min, pH value 5.5, dissolved oxygen amount 5%, the NaOH that neutralizing agent is 6mol/L, fermentation time 44h.After fermentation ends, the freeze-drying fermentation astragalus liquid Radix Astragali lyophilized powder that must ferment.Recording in the fermentation Radix Astragali massfraction of the Radix Astragali and be in 78%, 400g fermentation Radix Astragali lyophilized powder is 312g containing the quality of the Radix Astragali.
The liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 26.35 parts; Peptone: 2.20 parts; Glucose: 0.67 part; Yeast powder: 1.53 parts; 0.05 part, magnesium sulfate; 0.30 part of Triammonium citrate; 0.50 part of hydrogen sulfate dipotassium; 0.92 part of sodium acetate; Astragalus membranaceus powder: 90 parts; Water: 900 parts.
The Radix Astragali lyophilized powder that ferments in embodiment 2 is made an addition to according to 1% dosage in the broiler fodder of 21 ages in days and feed 28 days, after off-test, find that the body weight of fermentation Radix Astragali group broiler chicken is apparently higher than blank group and crude drug Radix Astragali group (P>0.05).
embodiment 3
The preparation method of the fermentation Radix Astragali of the present invention is:
After clean Radix Astragali crude drug medicine materical crude slice is pulverized, cross 20 mesh sieves, with the 10L ferment tank Radix Astragali, the bacterial concentration of fermentation strain LZMYFGM9 is 2 × 10
7individual/mL, connecing bacterium amount is that 4.5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 39 DEG C of temperature, rotating speed 200r/min, pH value 6.0, dissolved oxygen amount 8%, the NaOH that neutralizing agent is 6mol/L, fermentation time 52h.After fermentation ends, the freeze-drying fermentation astragalus liquid Radix Astragali lyophilized powder that must ferment.Recording in the fermentation Radix Astragali massfraction of the Radix Astragali and be in 78%, 400g fermentation Radix Astragali lyophilized powder is 312g containing the quality of the Radix Astragali.
The liquid nutrient medium of the fermentation Radix Astragali has been prepared by the composition of following parts by weight: whey powder: 24.00 parts; Peptone: 2.39 parts; Glucose: 0.50 part; Yeast powder: 1.68 parts; 0.03 part, magnesium sulfate; 0.50 part of Triammonium citrate; 0.30 part of hydrogen sulfate dipotassium; 1.10 parts of sodium acetates; Astragalus membranaceus powder: 100 parts; Water: 1100 parts.
The Radix Astragali lyophilized powder that ferments in embodiment 3 is made an addition to according to 1% dosage in the broiler fodder of 21 ages in days and feed 28 days, after off-test, find that the body weight of fermentation Radix Astragali group broiler chicken is apparently higher than blank group and crude drug Radix Astragali group (P>0.05).
embodiment 4
Macroporous adsorbent resin D101 and AB-8 used in the present embodiment are respectively 30.06mg/g and 30.58mg/g to the static adsorbance of fermentation Radix Astragali fluid extract saponin(e; Static adsorbance to crude drug Radix Astragali fluid extract saponin(e is respectively 32.40mg/g and 40.56mg/g; Dynamic adsorption amount to fermentation Radix Astragali fluid extract saponin(e is respectively 22.13mg/g and 26.25mg/g; Dynamic adsorption amount to crude drug Radix Astragali fluid extract is respectively 30.53mg/g and 32.62mg/g.
Macroporous adsorbent resin in the present embodiment, the AB-8 resin that every 5mL handles well and D 101 resins are equivalent to respectively dried resin 1.1g and 1.0g.
The extracting method of a kind of Radix Astragali total saponins that ferments provided by the invention, comprises the following steps:
(1) take fermentation Radix Astragali lyophilized powder 400g prepared by embodiment 1, cross 20 mesh sieves, with airtight immersion 8h under 20 times of amounts (i.e. 8000 mL), 85% ethanol room temperature, 85 DEG C of temperature are soaked 1h, filter, and filter residue repeats to extract 1 time again.Merge supernatant liquor twice, filter cleaner.The dregs of a decoction are used for extracting water miscible other effective constituent, as polysaccharide etc., are beneficial to abundant exploitation and efficient utilization of leavened prod effective constituent.Underpressure distillation alcohol extract reclaims ethanol, and (pumping pressure is 0.08MPa, bath temperature is 55 DEG C), obtain alcohol extracting fermentation Radix Astragali fluid extract 160g, 1g fluid extract is containing the 1.95g crude drug Radix Astragali, detect through vanillin-sulfuric acid method, in alcohol extracting fermentation Radix Astragali fluid extract, total saponin content is 11.48 %.
(2) with 100mL pure water dilution 10g alcohol extracting fermentation Radix Astragali fluid extract, filter, with total saponins in 100mL water-saturated n-butanol extraction filtrate 3 times, at the uniform velocity slight jolting in the same direction when extraction, after slightly static, then row slight jolting in the other direction, to avoid emulsion.
Merge three times butanol extraction liquid, wash propyl carbinol liquid with water 2 times, each 100mL.Merge propyl carbinol liquid, propyl carbinol is reclaimed in underpressure distillation, and (pumping pressure is 0.08MPa, and bath temperature is 60 DEG C～85 DEG C, it is 15%～30% water that distillation can slowly add volumn concentration at need), extremely, without propyl carbinol taste, extracting solution is concentrated into 21g, in 1g concentrated solution, contains the 0.93g crude drug Radix Astragali.Because of n-butyl alcohol extract not soluble in water after dry, therefore concentrated solution is prepared into 1:0.9～1.1, in 1g enriched material containing 0.9～1.1g crude drug Radix Astragali.
(3) with the n-butanol extraction concentrated solution of a small amount of anhydrous alcohol solution fermentation Radix Astragali, then with the dilution of 100mL pure water, filter, supernatant liquor is slowly splined on to AB-8 macroporous adsorptive resins (Φ 2.4cm × 70cm), standing 30min.Then use 1 times of column volume of 30% ethanol elution (300mL), abandon elutriant.Use again 3 times of column volumes of 85% ethanol elution (950mL).Mean flow rate is 5mL/min, collects elutriant, and ethanol is reclaimed in underpressure distillation, and lyophilize, obtains purification of fermentation Radix Astragali total saponins 3.05g, and in fermentation Radix Astragali purifying saponin(e, total saponin content is 88.39%.
embodiment 5
The extraction of crude drug Radix Astragali total saponins of the present invention comprises the following steps:
(1) take the crude drug Radix Astragali 312 g, cross 20 mesh sieves, with airtight immersion 8h under 20 times of amounts (6240 mL), 85 % ethanol room temperatures, 85 DEG C of temperature are soaked 1h, filter, and filter residue repeats to extract 1 time again.Merge supernatant liquor twice, filter cleaner.Underpressure distillation alcohol extract reclaims ethanol, and (pumping pressure is 0.08 MPa, bath temperature is 55 DEG C), obtain alcohol extracting crude drug Radix Astragali fluid extract 150 g(1 g fluid extracts containing the 2.08 g crude drug Radixs Astragali), detect through vanillin-sulfuric acid method, in alcohol extracting crude drug Radix Astragali fluid extract, total saponin content is 16.78 %.
(2) dilute 10 g alcohol extracting crude drug Radix Astragali fluid extracts with 100 mL pure water, filter, with total saponins in 100 mL water-saturated n-butanol extraction filtrates, 3 times (at the uniform velocity slight jolting in the same direction when extraction, after slightly static, row slight jolting in the other direction again, to avoid emulsion).
Merge three times butanol extraction liquid, wash propyl carbinol liquid with water 2 times, each 100 mL.Merge propyl carbinol liquid, propyl carbinol (pumping pressure is 0.08 MPa, and bath temperature is 60 DEG C～85 DEG C, and distillation can slowly add the water of 15 %～30 % at need) is reclaimed in underpressure distillation, extremely, without propyl carbinol taste, extracting solution is concentrated in 19 g(1 g concentrated solutions containing the 1.09 g crude drug Radixs Astragali).
(3) with the n-butanol extraction concentrated solution of a small amount of anhydrous alcohol solution crude drug Radix Astragali, then with 100 mL pure water dilutions, filter, supernatant liquor is slowly splined on to AB-8 macroporous adsorptive resins (Φ 2.4 cm × 70 cm), leave standstill 30 min.Then use 1 times of column volume of 30 % ethanol elution (300 mL), abandon elutriant.Use again 3 times of column volumes of 85 % ethanol elution (950 mL).Mean flow rate is 5 mL/min, collects elutriant, and ethanol is reclaimed in underpressure distillation, and lyophilize, obtains purifying crude drug Radix Astragali total saponins 3.92 g, detects through vanillin-sulfuric acid method, and in crude drug Radix Astragali purifying saponin(e, total saponin content is 91.27 %.
The fermentation Radix Astragali total saponins content that use the inventive method is extracted and yield, all lower than crude drug Radix Astragali total saponins, illustrate that obvious variation has occurred saponins material in the biotransformation of the Radix Astragali.The total saponin that the inventive method is extracted and content is all higher than using separately n-butanol extraction or macroporous resin enrichment method, and the inventive method operation is comparatively easy, be easy to promote and expanding production.
embodiment 6
The difference of the present embodiment and embodiment 4 is:
The fermentation Radix Astragali lyophilized powder that the present embodiment adopts the preparation method in embodiment 1 to prepare.
In step (3), with the n-butanol extraction concentrated solution of a small amount of anhydrous alcohol solution fermentation Radix Astragali, then with the dilution of 100mL pure water, filter, supernatant liquor is slowly splined on to AB-8 macroporous adsorptive resins (Φ 2.4cm × 70cm), leave standstill 30min.Then use 1.1 times of column volumes of 30% ethanol elution (330mL), abandon elutriant.Use again 6 times of column volumes of 70% ethanol elution (1800mL).Mean flow rate is 5mL/min, collects elutriant, and ethanol is reclaimed in underpressure distillation, and lyophilize, obtains purification of fermentation Radix Astragali total saponins 2.96g, and in fermentation Radix Astragali purifying saponin(e, total saponin content is 78.52%.
All the other steps are all identical.
embodiment 7
The difference of the present embodiment and embodiment 4 is:
The fermentation Radix Astragali lyophilized powder that the present embodiment adopts the preparation method in embodiment 1 to prepare.
In step (3), with the n-butanol extraction concentrated solution of a small amount of anhydrous alcohol solution fermentation Radix Astragali, then with the dilution of 100mL pure water, filter, supernatant liquor is slowly splined on to AB-8 macroporous adsorptive resins (Φ 2.4cm × 70cm), leave standstill 30min.Then use 0.9 times of column volume of 30% ethanol elution (270mL), abandon elutriant.Use again 4 times of column volumes of 90% ethanol elution (1200mL).Mean flow rate is 5mL/min, collects elutriant, and ethanol is reclaimed in underpressure distillation, and lyophilize, obtains purification of fermentation Radix Astragali total saponins 3.24g, and in fermentation Radix Astragali purifying saponin(e, total saponin content is 84.81%.
All the other steps are all identical.
embodiment 8
The difference of the present embodiment and embodiment 4 is: the macroporous adsorbent resin adopting in the present embodiment is D101.Obtain purification of fermentation Radix Astragali total saponins 2.83g, in fermentation Radix Astragali purifying saponin(e, total saponin content is 86.39%.
All the other steps are all identical.
embodiment 9
The present embodiment has been investigated 6 kinds of macropores and has been set the enriching and purifying ability to fermentation Radix Astragali total saponins, by quality and the total saponin content of the Radix Astragali total saponins that relatively ferments, optimize AB-8 and D101 effect is better, nonpolar and macroporous adsorbent resin low-pole be relatively suitable for fermenting enrichment and the purifying of Radix Astragali saponin are described.Method and the step of test the results are shown in Table 1 with the comparison test of 4,6 kinds of macroporous resins of embodiment.
Richness and the purification experiment result of 6 kinds of macroporous resins of table 1 to fermentation Radix Astragali saponin
。
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. the ferment preparation method of the Radix Astragali, is characterized in that: step is as follows: after clean Radix Astragali crude drug medicine materical crude slice is pulverized, and the fermentation Radix Astragali, the bacterial concentration of the non-solution lactose of fermentation strain suis CGMCC No.4227 is 2 × 10
6～2 × 10
8individual/mL, connecing bacterium amount is that 4%～5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 38 DEG C～39 DEG C of temperature, rotating speed 200r/min, pH value 5.5～6.5, dissolved oxygen amount 5%～10%, neutralizing agent is the NaOH of 6mol/L, fermentation time 44h～52h, after fermentation ends, the freeze-drying fermentation astragalus liquid Radix Astragali product that must ferment; Preferably, the bacterial concentration of fermentation strain CGMCC No.4227 is 2 × 10
8individual/mL, connecing bacterium amount is that 5%(accounts for fermentation cumulative volume), Radix Astragali consumption is 8%(Radix Astragali quality: fermentating liquid volume, g:mL), 39 DEG C of temperature, rotating speed 200r/min, pH value 6.4, dissolved oxygen amount 10%, the NaOH that neutralizing agent is 6mol/L, fermentation time 48h; Preferably, the liquid nutrient medium using in described fermentation Radix Astragali process has been prepared by the composition of following parts by weight: whey powder: 24～27 parts; Peptone: 2.2～2.4 parts; Glucose: 0.5～0.7 part; Yeast powder: 1.4～1.7 parts; Magnesium sulfate: 0.03～0.05 part; Triammonium citrate: 0.30～0.50g; Hydrogen sulfate dipotassium: 0.30～0.50g; Sodium acetate: 0.90～1.10g; Astragalus membranaceus powder: 80～100 parts; Water: 900～1100 parts; More preferably, the liquid nutrient medium using in described fermentation Radix Astragali process has been prepared by the composition of following parts by weight: whey powder: 25.79 parts; Peptone: 2.28 parts; Glucose: 0.60 part; Yeast powder: 1.56 parts; Magnesium sulfate: 0.04 part; Triammonium citrate: 0.40 part; Hydrogen sulfate dipotassium: 0.40 part; Sodium acetate: 1.00 parts; Astragalus membranaceus powder: 80 parts; Water: 1000 parts.
2. the fermentation Radix Astragali claimed in claim 1 is as the application of fodder additives.
3. a fodder additives, is characterized in that: the effective constituent of described fodder additives is the fermentation Radix Astragali claimed in claim 1.
4. the ferment extracting method of Radix Astragali total saponins, is characterized in that: step is as follows:
(1) the fermentation Radix Astragali claimed in claim 1 is got with 80%～90% ethanol temperature lixiviate, preferably got with 85% ethanol temperature lixiviate, obtain alcohol extracting fermentation Radix Astragali fluid extract; Preferably, in described fluid extract, 1g fluid extract contains 0.9～1.1g crude drug Radix Astragali;
(2) alcohol extracting fermentation Radix Astragali fluid extract water saturation n-butanol extraction step (1) being obtained, obtains extracting solution;
(3) extracting solution is splined on to macroporous adsorptive resins absorption, wash-out, collects elutriant, and elutriant is carried out to aftertreatment, obtains the fermentation Radix Astragali total saponins after purifying.
5. extracting method according to claim 4, is characterized in that: it is that 83 DEG C～86 DEG C temperature are soaked 1h～2h by airtight immersion 6h～10h under 80%～90% ethanol room temperature for the fermentation Radix Astragali that described temperature lixiviate is got, and filters, and filter residue repeats to extract 1 time again; Preferably, it is that 85 DEG C of temperature are soaked 1h by airtight immersion 8h under 85% ethanol room temperature for the fermentation Radix Astragali that described temperature lixiviate is got, and filters, and filter residue repeats to extract 1 time again.
6. extracting method according to claim 4, is characterized in that: when described temperature lixiviate is got, every 1g fermentation Radix Astragali 20ml 85% ethanol temperature lixiviate is got.
7. extracting method according to claim 4, is characterized in that: while using water saturation n-butanol extraction in step (2), and at the uniform velocity slight jolting in the same direction, after slightly static, slighter jolting in the other direction.
8. extracting method according to claim 4, is characterized in that: described in step (3), macroporous adsorbent resin is AB-8 or D101; Be preferably AB-8.
9. extracting method according to claim 4, is characterized in that: described in step (3), wash-out is first to use 30% ethanol elution, abandons elutriant; Use again 70%～90% ethanol elution, collect elutriant; Preferably, described wash-out is first to use 30% ethanol elution, abandons elutriant; Use again 85% ethanol elution, collect elutriant.
10. according to the arbitrary described extracting method of claim 4-9, it is characterized in that: after extracting, elutriant is after concentrate drying, and fermentation Radix Astragali total saponins content reaches 78.52%～88.39%.
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